Humans are infected by two herpes simplex viruses respectively designated HSV-1 and HSV-2. The two viruses have well defined biologic and antigenic differences but are 50% similar at the DNA and protein levels (have 50% type common antigenic determinants). There are 78 proteins which are encoded by the viral DNAs. In most of these, approximately 50% of the antigenic determinants are type-common and they are generally the most antigenic. Only a few proteins have primarily type-specific epitopes.
Mucosal and skin surfaces (generally abraded skin) are the usual site of infection with both of these viruses. The mouth and lips are the most common sites of infection with HSV-1, while HSV-2 is associated with genital infections. Although HSV-1 and HSV-2 are usually transmitted by different routes and involve different areas of the body, there is much overlap between the epidemiology and clinical manifestations of infections caused by these viruses.
Primary infection with HSV-1 generally occurs in children less than 5 years of age where it is often asymptomatic or very mild manifesting as oropharyngeal disease (gingivostomatitis). Primary infection can also occur in young adults where it has been associated with pharyngitis and, often, a mononucleosis-like syndrome. Primary HSV-1 infection leads to virus shedding in the mouth for as long as 23 days, on the average for 7-10 days. Neutralizing antibodies begin to appear on days 4-7 after clinical onset of disease and peak at approximately 3 weeks. Neutralizing antibodies and antibody-dependent cellular cytotoxicity antibodies persist for the lifetime of the host. Virus shedding in the saliva of asymptomatic children was documented in approximately 3-20% of children under 15 years of age. Virus is transmitted from infected to susceptible individuals by respiratory droplets or through direct contact with infected secretions during close personal contact. Primary infection is followed by the establishment of latency with periodic recurrent symptoms. Primary infection with HSV-2 occurs later in life (transmission is generally by sexual contact) and it occurs in the presence of neutralizing antibody to HSV-1. The presence of HSV-1 antibodies prior to infection with HSV-2 generally affects the development of the HSV-2 antibodies.
Studies designed to examine the protein specificity of the antibody response have shown that early after infection antibodies appear to gD, gB, ICP4, gE, gG and gC although not all proteins were studied. The more severe the primary infection or the more frequent the recurrences, the greater the band intensity or the quantity of antibodies. However, the absence of an antibody response (or the enhanced antibody response) to a specific viral protein does not correlate with the severity of the disease or the frequency of recurrences. In fact, the appearance of antibody to a specific protein depends on the inherent antigenicity of the specific protein (as determined after exposure only to that protein) and its presentation to the immune system by professional antigen presenting cells. The levels of expression of the specific protein in virus infected cells is effected by the presence of additional viral proteins. The appearance of antibody to a specific viral protein is further regulated by the previous immune status of the host. For example, antibodies to HSV-2 proteins must develop in a host that already has antibody to the homologous HSV-1 proteins resulting, for at least some of the HSV-2 proteins, in downregulation.
Serologic Assays
A variety of serologic assays are available, although none is currently practical for quantitating type-specific antibodies. Indeed, many assays can give rise to confound the results. Because of the extensive cross-reactivity between the two HSV serotypes, it is difficult to detect HSV-2 antibodies in patients who already have high titers of antibodies to HSV-1 and vice versa. Therefore serodiagnosis is not routinely done in diagnostic laboratories. Available assays include neutralization, complement fixation, passive hemagglutination, antibody-dependent cytotoxicity, ELISA and immunoblots.
Microneutralization, originally used to distinguish between antibody to HSV-1 and HSV-2, has since been shown to suffer from problems due to the presence of type-common antibodies. Another variation on the microneutralization, termed the multiplicity analysis kinetics of neutralization assay (MAKNA) was more specific because it used an artificial mixture consisting of wild type HSV-2 and a HSV-1 mutant which lacks a viral glycoprotein designated gC that primarily induces type specific antibody (Infection and Immunity 40:184 (1983)). The virus mixture used in the MAKNA assay consists of type-common and type-specific antigens. However, the assay is not widely accepted for clinical use because it is very complex and can only be performed in research laboratories.
Recently, immunoblot assays using gG-1/gG-2 have been described as the method of choice for the detection of type specific assays. However, there is a significant body of data which indicate that gG assays have low sensitivity. Thus, in direct comparison experiments, the effectiveness of glycoproteins to induce high titers of serum antibody was ranked as: gD&gt;gB&gt;gI&gt;(gC=gE)&gt;gG&gt;gH (Invest. Ophthalmol. Visual Sci., 36:1352 (1995); J. Virol., 68:2118 (1994)). In a large scale study, among women with gG-2 antibody (measured by immunodot) only 13% reported a history of genital herpes; among men with gG-2 antibody only 19% reported a history of genital herpes, while most of those patients had HSV-2 antibodies. A similar pattern of low sensitivity and moderate specificity was also seen for facial herpes infection and gG-1 antibody and probably reflects the relatively poor antigenicity of gG (J. Am. Med. Assoc., 268:1702 (1992)). Anti-gG-2 antibody was detected only in 47% of cases with HSV-2 infection after a prior HSV-1 infection suggesting that a prior HSV-1 infection modifies the HSV type-specific serological response (J. Gen. Virol., 70:2365 (1989)). Major problems with identification of patients with HSV-1 antibody or dual antibody status using gG was also shown by others (J. Virol. Methods, 18:159 (1987)).
It is, therefore evident that a serologic assay capable of differentiating between HSV-1 and HSV-2 antibody while retaining good sensitivity is not yet available. The methods currently in use in the art lack sensitivity and accuracy. The instant invention solves this problem and provides a highly specific assay using a patient's blood to distinguish HSV-1 antibodies from HSV-2 antibodies.